Difference between revisions of "Part:BBa K2324006"

Latest revision as of 21:44, 1 November 2017

pRha_FimH_225sfGFP

The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 225 of the mature FimH protein.

This part produces a FimH adhesin protein fused with sfGFP (Pedelacq et al 2005) at its 225th amino acid residue, after cleavage of the signal peptide. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, should produce a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the fim operon. Unfortunately despite best efforts (multiple growth temperatures and multiple rhamnose inducing concentrations) no fluorescence has been detected in any of the E. coli strains.

References

Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.

Pallesen, L., Poulsen, L. K., Christiansen, G., and Klemm, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.

Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88.

Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 451
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K2324006 short</partinfo>
+<p>
-This part synthesises a FimH adhesin protein fused with GFP at its 225th amino acid residue. The coding sequence
+The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 225 of the mature FimH protein.</p>
+<p>
+This part produces a FimH adhesin protein fused with sfGFP (Pedelacq et al 2005) at its 225th amino acid residue, after cleavage of the signal peptide. The coding sequence
 is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced,
-produces a fluorescent FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid
+should produce a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid
-containing the <i> fim operon </i>.
+containing the <i> fim</i> operon. Unfortunately despite best efforts (multiple growth temperatures and multiple rhamnose inducing concentrations) no fluorescence has been detected in any of the <i>E. coli</i> strains.</p>
+<h2>References </h2>
+Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.
+Pallesen, L., Poulsen, L. K., Christiansen, G., and Klemm, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.
+Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88.
+Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.
 <!-- Add more about the biology of this part here